© 2000 American Heart Association, Inc.
Integrative Physiology |
Gene Transfer of Calcitonin Gene–Related Peptide Prevents Vasoconstriction After Subarachnoid Hemorrhage
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—We sought to determine whether adenovirus-mediated gene transfer in vivo of calcitonin gene–related peptide (CGRP), a potent vasodilator, ameliorates cerebral vasoconstriction after experimental subarachnoid hemorrhage (SAH). Arterial blood was injected into the cisterna magna of rabbits to mimic SAH 5 days after injection of AdRSVCGRP (8x108 pfu), AdRSVßgal (control virus), or vehicle. After injection of AdRSVCGRP, there was a 400-fold increase in CGRP in cerebrospinal fluid. Contraction of the basilar artery to serotonin in vitro was greater in rabbits after SAH than after injection of artificial cerebrospinal fluid (P<0.001). Contraction to serotonin was less in rabbits with SAH after AdRSVCGRP than after AdRSVßgal or vehicle (P<0.02). Basal diameter of the basilar artery before SAH (measured with digital subtraction angiogram) was 13% greater in rabbits treated with AdRSVCGRP than in rabbits treated with vehicle or AdRSVßgal (P<0.005). In rabbits treated with vehicle or AdRSVßgal, arterial diameter after SAH was 25±3% smaller than before SAH (P<0.0005). In rabbits treated with AdRSVCGRP, arterial diameter was similar before and after SAH and was reduced by 19±3% (P<0.01) after intracisternal injection of CGRP-(8-37) (0.5 nmol/kg), a CGRP1 receptor antagonist. To determine whether gene transfer of CGRP after SAH may prevent cerebral vasoconstriction, we constructed a virus with a cytomegalovirus (CMV) promoter, which results in rapid expression of the transgene product. Treatment of rabbits with AdCMVCGRP after experimental SAH prevented constriction of the basilar artery 2 days after SAH. Thus, gene transfer of CGRP prevents cerebral vasoconstriction in vivo after experimental SAH.
Key Words: gene therapy • cerebral vasospasm • vasodilation • neuropeptide • rabbit
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cerebral vasospasm is a delayed, prolonged arterial constriction that follows subarachnoid hemorrhage (SAH), which can lead to brain ischemia and infarction.1 Vasospasm occurs in 30% to 40% of patients after SAH1 and is the leading cause of mortality (28%) and morbidity (39%) in patients after SAH.2 Several mechanisms may contribute to vasospasm after SAH, including impaired endothelium-dependent relaxation; production of endothelium-derived contracting factors, including endothelin; and impaired activity of potassium channels in cerebral blood vessels.3 Cerebral vascular muscle is depolarized after SAH, most likely as a result of inhibition of potassium channels, and this depolarization is thought to contribute to vasospasm.4 Although several dilator mechanisms, including NO and nitrovasodilators, are impaired after experimental SAH in most studies,5 6 7 dilation of large cerebral arteries is not impaired in response to synthetic (aprikalim and cromakalim) and endogenous (calcitonin gene-related peptide [CGRP]) openers of potassium channels.6 7 8 Thus, we speculated that openers of potassium channels in vascular muscle may prevent vasospasm after SAH.
CGRP is an extremely potent vasodilator, which hyperpolarizes arterial muscle at least in part via opening of potassium channels.9 10 The peptide is abundant in perivascular nerve fibers surrounding cerebral blood vessels,11 and depletion of CGRP may have functional effects in the cerebral circulation after SAH.12 13 Intrathecal and intravenous injection of exogenous CGRP increases arterial diameter in animals with vasospasm after experimental SAH.14 15 16 Thus, it seemed likely that, if a sufficient amount of CGRP is released around cerebral arteries after SAH, vasospasm might be prevented.
Gene transfer to vascular adventitia via cerebrospinal fluid (CSF) is an appealing strategy to modulate cerebral vascular function.17 18 We made a recombinant adenovirus that encodes prepro-CGRP and reported that gene transfer via CSF in normal rabbits expresses biologically active CGRP, increases the level of cAMP (a second messenger for the vasodilator response to CGRP) in the basilar artery, and attenuates contraction of the artery in vitro.17 Exposure of canine cerebral arteries to blood in vivo does not diminish, or may even augment, efficiency of expression after gene transfer in vivo19 and in vitro.20 Thus, we speculated that perivascular application in vivo of a virus that expresses CGRP might prevent constriction of cerebral arteries after SAH. The goal of this study was to test the hypothesis that gene transfer of CGRP in vivo ameliorates cerebral vasoconstriction after SAH.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We made a replication-deficient adenovirus encoding prepro-CGRP (AdCGRP) or ß-galactosidase (Adßgal) with a Rous sarcoma virus (RSV) promoter.17 We also constructed an adenovirus with a cytomegalovirus (CMV) promoter encoding prepro-CGRP (AdCMVCGRP) and used AdCMVßgal. Male New Zealand White rabbits weighing 2.4 to 3.2 kg were anesthetized intramuscularly with xylazine and ketamine. A needle was inserted into the cisterna magna, 250 µL of CSF was withdrawn, and 250 µL of viral suspension (PBS with 3% sucrose with 3x109 pfu/mL virus) or vehicle (PBS with 3% sucrose) was infused.17
Experimental SAH was produced in rabbits 5 days after injection of virus (AdRSVCGRP or AdRSVßgal) or vehicle. The anesthetized rabbit was placed in a prone position, and a needle was inserted into the cisterna magna. After withdrawal of 1 mL of CSF, 1 mL/kg of fresh, nonheparinized autologous arterial blood was injected.7 16 21
Two days after experimental SAH (day 7), rabbits were anesthetized with pentobarbital IV and exsanguinated. Basilar arteries were removed and cut into rings (3 mm in length) for recording of isometric tension to examine vascular reactivity.17 Contraction to KCl, serotonin, and histamine was examined. Contraction was expressed as a percentage of response to 40 mmol/L KCl. Relaxation to acetylcholine and sodium nitroprusside was examined after vasocontraction with 1 to 3 µmol/L of histamine. Relaxation was expressed as percentage of precontraction. In separate experiments, arterial rings were examined for relaxation to synthetic CGRP 2 days after injection of arterial blood or artificial CSF (without gene transfer).
Digital subtraction angiography was performed immediately before and 2 days after experimental SAH in rabbits treated with virus. The anesthetized rabbit was placed in a supine position, and an angiocatheter was introduced through the exposed femoral artery into the thoracic aorta to measure systemic blood pressure. Then the catheter was advanced into the left vertebral artery, and angiography was performed after injection of nonionic contrast medium through the catheter.16 21 22 23 In some rabbits, synthetic CGRP or CGRP-(8-37) was injected into the cisterna magna immediately after the second angiogram, and angiography was repeated 15 minutes later. The basilar artery was divided into 3 segments of equal length, and the diameter at the midpoint of each segment was averaged for the final value. We used different rabbits for angiography and studies of vascular reactivity in vitro.
In other studies, we injected vehicle or virus into the cisterna magna 30 minutes after experimental SAH. We injected 4 rabbits with vehicle, 4 rabbits with AdCMVßgal, and 8 rabbits with AdCMVCGRP. Angiography was performed before and 2 days after SAH. The goal was to determine whether gene transfer of CGRP after, as well as before, SAH can be used to prevent vasoconstriction.
We collected CSF before injection of virus, just before SAH, and 2 days after SAH and measured CGRP immunoreactivity in CSF by RIA.17 In separate experiments, we measured CGRP immunoreactivity in CSF from rabbits after experimental SAH without gene transfer, to determine effects of SAH on release of CGRP. CGRP immunoreactivity in CSF was also measured in rabbits after treatment with AdRSVCGRP, without SAH.
Histochemistry for ß-galactosidase was performed 7 days after injection of AdRSVßgal.17 24
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effects of SAH or Gene Transfer on Release of CGRP Into CSF
Before studying rabbits after experimental SAH treated with adenoviral vectors, we performed experiments to determine effects of experimental SAH or gene transfer alone on expression of CGRP in CSF. CGRP immunoreactivity was measured in CSF from rabbits after injection of blood into CSF without gene transfer (Figure 1A
). Peak concentration of CGRP 30 minutes after
injection of blood was 0.17±0.10 (mean±SEM) nmol/L. Concentration of
CGRP in serum from the same rabbits was 0.44±0.09 nmol/L.
|
CGRP concentration was measured in CSF from rabbits treated
with AdRSVCGRP without experimental SAH
(Figure 1B
). Between 3 and 7 days after injection of the
virus, concentration of CGRP exceeded 1 nmol/L, with peak expression of
CGRP 5 days after injection of the virus (2.2±0.9 nmol/L). This course
of expression of the transgene product in CSF was similar to the
previous finding of expression in other tissues after injection of
virus with the RSV
promoter.25 On the
basis of this result, we injected blood into the cisterna magna of
rabbits 5 days after injection of virus in the following
study.
Gene Transfer In Vivo
We injected adenovirus into CSF of rabbits, then
injected blood to mimic SAH 5 days later (day 5), and performed final
assays 2 days later (day 7). All rabbits tolerated injection of
adenovirus without detectable neurological deficits. Immediately after
injection of blood, almost all rabbits developed transient apnea. Seven
of 53 rabbits (13%) died within a few minutes after injection of
blood. Data from those rabbits were excluded. Rectal temperature was
39.3±0.1°C before injection of virus, 39.4±0.1°C before injection
of blood (day 5), and 39.4±0.1°C on day 7. Leukocyte count in CSF
increased from 3±1/mm3 to
327±59/mm3 5 days after injection of
AdRSVßgal (before injection of blood) and
417±76/mm3 5 days after AdRSVCGRP. We did
not measure leukocyte count on day 7, because contamination with blood
affected the counts.
In rabbits treated with AdRSVßgal or vehicle, baseline
concentration of CGRP on day 0 was minimal (0.006±0.003 nmol/L), and
concentration did not increase on day 5 or day 7
(Figure 1C). In contrast, in rabbits treated with AdRSVCGRP,
the concentrations of CGRP in CSF on day 5 (1.9±0.2 nmol/L) and day 7
(2.0±0.3 nmol/L) were almost 400-fold greater than baseline
concentration (0.005±0.003 nmol/L), which indicates marked expression
of the transgene product.
With histochemistry, there was extensive staining for ß-galactosidase on the ventral surface of the brain from rabbits treated with AdRSVßgal and blood injection into CSF, as we previously demonstrated in other species.19 24 26 Thus, widespread overexpression of CGRP was also expected on the surface of brain after gene transfer of CGRP with experimental SAH.
Effect of Gene Transfer on Vasocontraction In
Vitro After Experimental SAH
Contraction to 40 mmol/L KCl was similar in arteries
exposed to blood and treated with vehicle (1.48±0.14 g), AdRSVßgal
(1.40±0.11 g), and AdRSVCGRP (1.50±0.12 g), and in arteries from
rabbits that received an injection of artificial CSF (instead of blood)
into the cisterna magna and treated with vehicle (control rabbits,
1.58±0.23 g).
The contractile response to serotonin (0.01 to 3 µmol/L)
was greatly augmented in arteries from rabbits after experimental SAH
that were treated with vehicle, compared with control rabbits without
SAH (P<0.001 for the entire concentration-response
curve; maximal response
[Rmax] was 98±7% versus
48±11%, P<0.0005,
Figure 2A). Contraction was less after AdRSVCGRP
(Rmax 70±11%) than after
vehicle or AdRSVßgal (Rmax
93±8%) (P<0.02 for entire curve;
P<0.05 for
Rmax).
|
P<0.05 vs
Adßgal; 
P<0.05 vs AdCGRP. E, Response to
synthetic CGRP of the histamine-precontracted basilar artery from
rabbits 2 days after injection with arterial blood (SAH) or aCSF.
Values are
mean±SEM.
Contractile responses to histamine (0.03 to 30 µmol/L)
were similar in arteries from rabbits after experimental SAH (treated
with vehicle) and from control rabbits without SAH
(Figure 2B). In arteries treated with AdRSVCGRP, contraction
to 3 and 10 µmol/L histamine was less than after vehicle or
AdRSVßgal (P<0.05).
Relaxation to high concentrations of acetylcholine (1 to 10
µmol/L) were attenuated after SAH (in rabbits treated with vehicle)
compared with control rabbits
(Rmax, 78±8% versus 99±1%,
P<0.05,
Figure 2C). After SAH, relaxation to acetylcholine did not
change after AdRSVCGRP compared with vehicle or AdRSVßgal. Relaxation
to nitroprusside was similar among the 4 groups
(Figure 2D).
In separate experiments, relaxation to synthetic CGRP (0.03
to 10 nmol/L) was virtually identical between the arteries from rabbits
2 days after experimental SAH and after injection of artificial CSF
instead of blood (without gene transfer,
Figure 2E).
Thus, vasocontraction to serotonin, which was augmented after SAH, and vasocontraction to histamine, which was not augmented, were both attenuated by gene transfer of CGRP. The response to synthetic CGRP was preserved after SAH.
Effect of Gene Transfer on Vasoconstriction In
Vivo After Experimental SAH
We tested the hypothesis that gene transfer of CGRP
prevents constriction of arteries in vivo. First, we determined effects
on resting diameter of the basilar artery 5 days after gene transfer
(Figure 3A). Injection of AdRSVßgal did not change diameter
on day 5 (899±19 µm) compared with vehicle (887±13 µm). In
rabbits treated with AdRSVCGRP, diameter on day 5 (1000±25 µm) was
13% greater than in rabbits treated with vehicle or AdRSVßgal
(P<0.005). Thus, gene transfer of CGRP increased
resting diameter of the basilar artery.
|
Arterial diameter was compared on day 7 (2 days after SAH)
and on day 5. In rabbits treated with vehicle, diameter was 25±3%
smaller on day 7 (670±28 µm) than on day 5
(P<0.001,
Figures 3A, 4A, and 4B). In rabbits treated with AdRSVßgal,
diameter was 23±3% smaller on day 7 (696±31 µm) than on day 5
(P<0.001). Vertebral arteries and vessels of the
circle of Willis, in addition to the basilar artery, were constricted
on day 7
(Figures 4A and 4B). In rabbits treated with AdRSVCGRP,
diameter was similar on days 5 and 7 (963±10 µm,
P>0.1,
Figures 3A, 4D, and 4E).
|
After intracisternal injection of CGRP (0.1 nmol/kg) to
rabbits treated with vehicle on day 7, diameter of the constricted
basilar artery returned to resting values (1±3%, n=5,
Figure 4C). After intracisternal injection (0.5 nmol/kg) of
a CGRP1 receptor antagonist, CGRP-(8-37), to
rabbits treated with AdRSVCGRP on day 7, the basilar artery constricted
by 19±3% (n=5, P<0.01,
Figures 3B and 4F). These findings suggest that either gene
transfer of CGRP or an intracisternal bolus injection of synthetic CGRP
can prevent vasoconstriction after experimental SAH by an effect on
CGRP1 receptors.
Systemic arterial pressure and arterial blood gases during
angiography were not significantly different between treatment groups
or at the time of the 2 angiograms in each group
(Table).
Thus, these physiological factors did not account for differences in
arterial diameter.
|
In the studies described above, we examined effects of
treatment with AdRSVCGRP before SAH. In other studies, we examined
effects of gene transfer of CGRP after experimental SAH. For these
studies, we constructed a recombinant virus with a CMV promoter,
because maximal expression is much more rapid with a CMV than an RSV
promoter.26 In
rabbits treated with vehicle or AdCMVßgal, diameter of the basilar
artery was 21±2% smaller 2 days after than before SAH
(Figure 5, control). Reduction in diameter after SAH was
similar in rabbits treated with vehicle (-23±3%, n=4) or
AdCMVßgal (-18±2%, n=4, P>0.1). In rabbits
treated with AdCMVCGRP, diameter was similar before and 2 days after
SAH
(Figure 5). Systemic arterial pressure and arterial blood
gases during angiography were not significantly different between
treatment groups or at the time of the 2 angiograms in each group (data
not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This is the first successful use of gene transfer of a vasoactive protein or peptide to prevent cerebral vasoconstriction in vivo after experimental SAH. There are 2 major new findings. First, gene transfer of CGRP in vivo prevented excessive contraction of the basilar artery to serotonin in vitro after experimental SAH. Second, gene transfer of CGRP prevented constriction of the basilar artery in vivo after experimental SAH both when the virus was applied before SAH and after SAH. Thus, gene transfer of CGRP is a promising approach for prevention of vasospasm after SAH.
Gene Transfer to Cerebral Arteries
A unique implication of the present study is for gene
therapy for cerebrovascular disease. Although clinical trials for
patients with coronary and peripheral vascular disease are under
way,27 gene transfer
has not received extensive study for even animal models of
cerebrovascular diseases. A reason for this lag may be that
intravascular gene delivery, which is feasible for coronary and
peripheral arteries, has not yet been accomplished for intracranial
cerebral arteries, because intravascular approaches require
interruption of blood flow to the brain.
As an alternative approach, perivascular gene delivery via
CSF can be used for overexpression in the cerebral arteries of
vasoactive proteins and peptides, including
CGRP17 and
endothelial NO synthase
(eNOS).18 An
advantage of perivascular gene delivery by injection into CSF versus
intravascular delivery is that expression of transgene products is not
limited to small regions but is distributed over an expanded area.
Because multiple cerebral arteries both in anterior and posterior
circulations are at risk for vasospasm after SAH, perivascular gene
delivery may be more beneficial than intravascular delivery for
prevention of vasospasm. In a recent study, perivascular application of
liposomes with oligonucleotides with high affinity for nuclear
factor-
B to act as decoy DNA also prevented cerebral
vasoconstriction after experimental
SAH.21
Vasodilators in SAH
The early
release28 and
subsequent depletion of
CGRP12 13
in and around cerebral arteries after SAH suggest that endogenous CGRP
may be protective in the cerebral circulation after SAH and that
depletion of CGRP may contribute to vasoconstriction. The findings in
Figure 1A confirm a previous finding that, 30 minutes after
experimental SAH, CGRP concentration in CSF reached a
peak,28 possibly
from release of endogenous CGRP after SAH. Because the peak
concentration of CGRP in CSF in both the present and previous
studies28 was lower
than concentrations in serum, however, blood contamination may
contribute substantially to increases in CSF levels of CGRP.
Importantly, the increase in endogenous CGRP in CSF derived from SAH
was far less than the enormous increase in transduced CGRP after gene
transfer.
The present and previous findings in vitro indicate that relaxation of cerebral arteries to CGRP is similar with and without SAH.8 The findings are in contrast to the previous finding in vivo that dilatation of the basilar artery to CGRP after SAH is augmented as compared with arteries without SAH.6 One possible explanation for differences in findings in vivo and in vitro is that effects of SAH on arteries may be attenuated in vitro, because arteries are removed from clot remnants and products in CSF that may contain vasoconstrictor substances.
The present and previous findings indicate that endothelium-dependent vasorelaxation is impaired after SAH.5 6 7 Several mechanisms have been proposed to account for this vascular dysfunction after SAH, including a reduction in production or activity of NO, impairment of activation of soluble guanylate cyclase or production of cGMP, inhibitory effect of hemoglobin on NO, and adhesion and infiltration of leukocytes in cerebral arteries.3 Adenovirus encoding eNOS (AdeNOS) has been used extensively for gene transfer to blood vessels.18 29 30 AdeNOS is useful for improvement of impaired NO-mediated relaxation in vitro of arteries after experimental SAH.20 The virus, however, has not yet been shown to be effective in vivo in prevention of vasoconstriction after SAH.31 Differences in effects in vivo and in vitro may be due in part to absence of effects of hemoglobin on NO-mediated relaxation of arterial rings in vitro. A previous study in vitro supports this hypothesis; although relaxation of the rabbit basilar artery to acetylcholine was normal after SAH, it was greatly inhibited compared with the artery without SAH once oxyhemoglobin solution was added to the organ bath.32 Impaired responses to NO after SAH are a major reason that we used adenovirus that expresses CGRP for the present study in vivo instead of AdeNOS.
Vasoconstrictors in SAH
In this study, vasocontraction in vitro to serotonin
was potentiated, and vasocontraction to KCl and histamine was not
altered, after experimental SAH. Results were similar to those of
previous studies in vitro of the basilar artery from
rabbits.33 34 35 36
Serotonin has been used commonly to test vascular reactivity after SAH,
and previous studies demonstrated that contraction to serotonin was
increased 2-fold or more after
SAH.33 34 35 36
Denervation hypersensitivity of cerebral arteries to serotonin and
norepinephrine, from a marked reduction or alteration in perivascular
adrenergic nerves, may contribute to altered vascular response to
serotonin.37
In contrast, contraction of the rabbit basilar artery to KCl33 34 or histamine35 in vitro was not altered after experimental SAH. Contraction to histamine, however, was augmented in arteries after SAH when oxyhemoglobin was added to the organ bath.32 Thus, it is possible that, although SAH has the potential to alter vascular responses to histamine, augmented responses were not demonstrated in the present and previous35 studies in the organ bath because vasoconstrictor substances from clot remnants and products in CSF were not present in the bath.
After gene transfer of AdRSVCGRP, contraction of the basilar artery to serotonin and histamine in vitro was attenuated in the present study and in a previous study in normal vessels.17 Attenuation of contraction was mediated by CGRP1 receptors, because pretreatment with CGRP-(8-37), an antagonist for CGRP1 receptors, restored the response to normal.17 AdRSVCGRP did not alter responses to KCl, possibly because depolarization from KCl may prevent hyperpolarization from CGRP. Thus, the present findings of altered vascular reactivity in vitro strongly suggested that gene transfer of CGRP might prevent vasoconstriction in vivo after SAH. Because studies in the organ bath have important limitations described above, however, confirmation in vivo was essential.
Altered Vascular Tone After Gene
Transfer
The present angiographic findings demonstrated several
effects of gene transfer on tone of the basilar artery. First, arterial
diameter was similar after treatment with Adßgal and vehicle. Thus,
although leukocytosis in CSF demonstrated a substantial inflammatory
response after injection of adenovirus, the inflammation itself did not
alter arterial diameter before or after exposure to blood. Responses of
normal arteries to several stimuli in vitro in a previous
study17 and in
arteries after SAH in the present study also were virtually identical
in arteries treated with Adßgal and vehicle.
Second, gene transfer of CGRP increased resting diameter in vivo before SAH and prevented vasoconstriction after experimental SAH by an effect on CGRP1 receptors. Vasodilator effects of transduced CGRP were greater in constricted arteries on day 7 (44% increase in diameter compared with vehicle-treated rabbits) than in nonconstricted arteries on day 5 (13%). After SAH, because endogenous CGRP is depleted in cerebral arteries,12 13 sensitivity of the basilar artery to CGRP may be greater. This hypothesis is consistent with previous findings that dilatation of the basilar artery in vivo to CGRP after SAH is augmented as compared with arteries without SAH.6 Another possible reason for augmented dilator effects in constricted arteries may be that vessels that are submaximally constricted have greater capacity for dilation than do nonconstricted vessels.
Limitations of the Study
Vasospasm is a frequent and often fatal complication
after SAH in
patients.1 2
Novel strategies, including perhaps gene therapy, are needed to prevent
vasospasm. An important limitation of this study, however, is that
experimental SAH in rabbits does not replicate vasospasm after SAH in
humans. The rabbit is commonly used, however, for experimental SAH,
with intracisternal injection of autologous blood. Previous studies
reported constriction of the rabbit basilar artery by 12% to 35%
after injection of blood into
CSF.16 21 22 23 38 39
Thus, the present finding of 25% reduction in diameter is comparable
with those in previous studies. Constriction of cerebral arteries does
not produce signs of ischemia unless constriction is much greater
(perhaps 50% or more), at least in
patients,40 so it is
not clear whether the present strategy will also be useful for
prevention of symptomatic vasospasm.
Another difference between vasospasm in rabbits and patients is the interval after onset of SAH. Previous reports in rabbits demonstrated that both angiographic evidence of vasoconstriction22 23 and augmented vasocontraction to serotonin in vitro33 are maximal 2 days after SAH. In this study, we first pretreated rabbits with virus before injection of blood to match the peak of transgene expression with the peak of vasoconstriction after SAH, as in a previous study of gene transfer for brain ischemia.25 In application of this method in patients, it obviously will not be possible to pretreat patients with gene transfer before SAH. Thus, we constructed a recombinant virus with a CMV promoter, for which duration of 2 days seems to be appropriate for maximal transgene expression.26 The finding that AdCMVCGRP can prevent cerebral vasoconstriction when it is injected after SAH suggests that gene therapy may eventually be used for prevention of vasoconstriction when the virus is injected after SAH. Patients frequently develop vasospasm 4 to 14 days after SAH.1 Thus, it is possible that a virus with the RSV promoter may be preferable to the CMV promoter for use in patients.
The inflammatory response to this recombinant virus, demonstrated in this study by an increase in leukocytes in CSF, would prevent use in patients. To minimize unwanted inflammatory or immune responses, coadministration of immunosuppressant drugs41 or immunomodulatory proteins42 may be useful. Newer adenoviral vectors may be less immunogenic and thus applicable to human use.43
Implications
Intravenous administration of synthetic CGRP to
patients after SAH altered velocity of blood flow in cerebral arteries,
suggesting dilatation of spastic
arteries,44 and
appeared to improve neurological
outcome.45 46
The use of intravenous CGRP, however, is limited by pronounced
hypotension.45
Intracisternal gene transfer of CGRP is likely to be more useful than
intravenous administration, because local gene transfer may avoid
systemic effects of CGRP, and sustained release of CGRP is likely to be
required for efficacy. Thus, we suggest that gene transfer has
important potential advantages over usage of exogenous peptides for
prevention of vasospasm. Although there are limitations in the current
vectors and animal models, as discussed above, the present study
provides evidence, or perhaps proof of principle, that gene transfer
may be useful for prevention of vasospasm after
SAH.
|
Acknowledgments |
|---|
This work was supported by NIH Grants HL 14388, HL 16066, NS 24621, HL 62984, HL 14355, NS 37386, and DK 54759 and funds from the Veterans Administration. We thank Drs Andrew F. Russo and Hiroshi Nakane for their advice; Dr Beverly L. Davidson, Richard D. Anderson, and the University of Iowa Gene Transfer Vector Core for preparation of virus; Dr William T.C. Yuh, Michael Ryan, and Jacque Wertz for assistance with angiography; Pamela K. Tompkins for assistance with histochemistry; and Arlinda LaRose for typing the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Mohr JP, Kistler JP, Fink ME. Intracranial aneurysms. In: Barnett HJM, Mohr JP, Stein BM, Yatsu FM, eds. Stroke: Pathophysiology, Diagnosis, and Management. 2nd ed. New York, NY: Churchill Livingstone Inc; 1992:617–643.
2. Kassell NF, Torner JC, Haley EC Jr, Jane JA, Adams HP, Kongable GL. The international cooperative study on the timing of aneurysm surgery, 1: overall management results. J Neurosurg. 1990;73:18–36.[Medline] [Order article via Infotrieve]
3.
Faraci
FM, Heistad DD. Regulation of the cerebral circulation: role of
endothelium and potassium channels. Physiol Rev. 1998;78:53–97.
4. Harder DR, Dernbach P, Waters A. Possible cellular mechanism for cerebral vasospasm after experimental subarachnoid hemorrhage in the dog. J Clin Invest. 1987;80:875–880.
5.
Kim P,
Schini VB, Sundt TM, Vanhoutte PM. Reduced production of cGMP underlies
the loss of endothelium-dependent relaxations in the canine basilar
artery after subarachnoid hemorrhage. Circ Res. 1992;70:248–256.
6.
Sobey
CG, Heistad DD, Faraci FM. Effect of subarachnoid hemorrhage on
dilatation of rat basilar artery in vivo. Am J
Physiol. 1996;271:H126–H132.
7.
Zuccarello
M, Bonasso CL, Lewis AI, Sperelakis N, Rapoport RM. Relaxation of
subarachnoid hemorrhage-induced spasm of rabbit basilar artery by the
K+ channel activator cromakalim.
Stroke. 1996;27:311–316.
8.
Hongo K,
Tsukahara T, Kassel NF, Ogawa H. Effect of subarachnoid hemorrhage on
calcitonin gene-related peptide-induced relaxation in rabbit basilar
artery. Stroke. 1989;20:100–104.
9. Nelson MT, Huang Y, Brayden JE, Hescheler J, Standen NB. Arteriolar dilations in response to CGRP involve activation of K+ channels. Nature. 1990;344:770–773.[Medline] [Order article via Infotrieve]
10.
Kitazono
T, Heistad DD, Faraci FM. Role of ATP-sensitive K+ channels in
CGRP-induced dilatation of basilar artery in vivo. Am J
Physiol. 1993;265:H581–H585.
11.
McCulloch
J, Uddman R, Kingman TA, Edvinsson L. Calcitonin gene-related peptide:
functional role in cerebrovascular regulation. Proc Natl Acad
Sci U S A. 1986;83:5731–5735.
12. Nozaki K, Kikuchi H, Mizuno N. Changes of calcitonin gene-related peptide-like immunoreactivity in cerebrovascular nerve fibers in the dog after experimentally produced subarachnoid hemorrhage. Neurosci Lett. 1989;102:27–32.[Medline] [Order article via Infotrieve]
13. Edvinsson L, Ekman R, Jansen I, McCulloch J, Mortensen A, Uddman R. Reduced levels of calcitonin gene-related peptide-like immunoreactivity in human brain vessels after subarachnoid haemorrhage. Neurosci Lett. 1991;121:151–154.[Medline] [Order article via Infotrieve]
14. Nozaki K, Uemura Y, Okamoto S, Kikuchi H, Mizuno N. Relaxant effect of calcitonin gene-related peptide on cerebral arterial spasm induced by experimental subarachnoid hemorrhage in dogs. J Neurosurg. 1989;71:558–564.[Medline] [Order article via Infotrieve]
15. Toshima M, Kassell NF, Tanaka Y, Dougherty DA. Effect of intracisternal and intravenous calcitonin gene-related peptide on experimental cerebral vasospasm in rabbits. Acta Neurochir (Wien). 1992;119:134–138.[Medline] [Order article via Infotrieve]
16. Imaizumi S, Shimizu H, Ahmad I, Kaminuma T, Tajima M, Yoshimoto T. Effect of calcitonin gene-related peptide on delayed cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits. Surg Neurol. 1996;46:263–271.[Medline] [Order article via Infotrieve]
17.
Toyoda
K, Faraci FM, Russo AF, Davidson BL, Heistad DD. Gene transfer of
calcitonin gene-related peptide to cerebral arteries. Am J
Physiol. 2000;278:H586–H594.
18.
Chen
AFY, Jiang S-W, Crotty TB, Tsutsui M, Smith LA, O’Brien T, Katusic ZS.
Effects of in vivo adventitial expression of recombinant endothelial
nitric oxide synthase gene in cerebral arteries. Proc Natl Acad
Sci U S A. 1997;94:12568–12573.
19.
Muhonen
MG, Ooboshi H, Welsh MJ, Davidson BL, Heistad DD. Gene transfer to
cerebral blood vessels after subarachnoid hemorrhage.
Stroke. 1997;28:822–829.
20.
Onoue
H, Tsutsui M, Smith L, Stelter A, O’Brien T, Katusic ZS. Expression
and function of recombinant endothelial nitric oxide synthase gene in
canine basilar artery after experimental subarachnoid hemorrhage.
Stroke. 1998;29:1959–1966.
21.
Ono S,
Date I, Onoda K, Shiota T, Ohtomo T, Ninomiya Y, Asari S, Morishita R.
Decoy administration of NF-
B into the subarachnoid space for
cerebral angiopathy. Hum Gene Ther. 1998;9:1003–1011.[Medline]
[Order article via Infotrieve]
22. Yanamoto H, Kikuchi H, Okamoto S, Nozaki K. Preventive effect of synthetic serine protease inhibitor, FUT-175, on cerebral vasospasm in rabbits. Neurosurgery. 1992;30:351–357.[Medline] [Order article via Infotrieve]
23. Shishido T, Suzuki R, Qian L, Hirakawa K. The role of superoxide anions in the pathogenesis of cerebral vasospasm. Stroke. 1994;25:864–868.[Abstract]
24.
Ooboshi
H, Welsh MJ, Rios CD, Davidson BL, Heistad DD. Adenovirus-mediated gene
transfer in vivo to cerebral blood vessels and perivascular tissue.
Circ Res. 1995;77:7–13.
25. Yang G-Y, Zhao Y-J, Davidson BL, Betz AL. Overexpression of interleukin-1 receptor antagonist in the mouse brain reduces ischemic brain injury. Brain Res. 1997;751:181–188.[Medline] [Order article via Infotrieve]
26.
Christenson
SD, Lake KD, Ooboshi H, Faraci FM, Davidson BL, Heistad DD.
Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and
perivascular tissue in mice. Stroke. 1998;29:1411–1416.
27.
Baumgartner
I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, Isner JM.
Constitutive expression of phVEGF165 after intramuscular gene transfer
promotes collateral vessel development in patients with critical limb
ischemia. Circulation. 1998;97:1114–1123.
28. Tran Dinh YR, Debdi M, Couraud J-Y, Creminon C, Seylaz J, Sercombe R. Time course of variations in rabbit cerebrospinal fluid levels of calcitonin gene-related peptide- and substance P-like immunoreactivity in experimental subarachnoid hemorrhage. Stroke. 1994;25:160–164.[Abstract]
29.
Von
der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M,
Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibiting neointimal
vascular lesion: in vivo transfer of endothelial cell nitric oxide
synthase gene. Proc Natl Acad Sci
U S A. 1995;92:1137–1141.
30.
Ooboshi
H, Chu Y, Rios CD, Faraci FM, Davidson BL, Heistad DD. Altered vascular
function after adenovirus-mediated overexpression of endothelial nitric
oxide synthase. Am J Physiol. 1997;273:H265–H270.
31. Macdonald RL, Ghadge G, Weihl C, Stoodley M, Johns L, Lin G, Kovalczuk A, Roos R. Effect of adenoviral nitric oxide synthase gene transfer on vasospasm after subarachnoid hemorrhage. Abstr Am Soc Gene Ther. 1998;154a. Abstract.
32. Tran Dinh Y-R, Roche S, Debdi M, Seylaz J, Sercombe R. Effects of oxyhemoglobin in vitro in cerebral arteries from normal animals and animals subject to subarachnoid hemorrhage or indomethacin treatment. Brain Res. 1998;790:91–97.[Medline] [Order article via Infotrieve]
33. Nakagomi T, Kassell NF, Sasaki T, Fujiwara S, Lehman RM, Johshita H, Nazar GB, Torner JC. Effect of subarachnoid hemorrhage on endothelium-dependent vasodilation. J Neurosurg. 1987;66:915–923.[Medline] [Order article via Infotrieve]
34. Vollmer DG, Takayasu M, Dacey RG. An in vitro comparative study of conducting vessels and penetrating arterioles after experimental subarachnoid hemorrhage in the rabbit. J Neurosurg. 1992;77:113–119.[Medline] [Order article via Infotrieve]
35.
Debdi
M, Seylaz J, Sercombe R. Early changes in rabbit cerebral artery
reactivity after subarachnoid hemorrhage. Stroke. 1992;23:1154–1162.
36. Sutter B, Suzuki S, Arthur AS, Kassell NF, Lee KS. Effects of subarachnoid hemorrhage on vascular responses to calcitonin gene-related peptide and its related second messengers. J Neurosurg. 1995;83:516–521.[Medline] [Order article via Infotrieve]
37. Lobato RD, Marín J, Salaices M, Rivilla F, Burgos J. Cerebrovascular reactivity to noradrenaline and serotonin following experimental subarachnoid hemorrhage. J Neurosurg. 1980;53:480–485.[Medline] [Order article via Infotrieve]
38. Diringer MN, Kirsch JR, Traystman RJ. Reduced cerebral blood flow but intact reactivity to hypercarbia and hypoxia following subarachnoid hemorrhage in rabbits. J Cereb Blood Flow Metab. 1994;14:59–63.[Medline] [Order article via Infotrieve]
39.
Nomura
H, Hirashima Y, Endo S, Takaku A. Anticardiolipin antibody aggravates
cerebral vasospasm after subarachnoid hemorrhage in rabbits.
Stroke. 1998;29:1014–1019.
40.
Simeone
FA, Trepper P. Cerebral vasospasm with infarction.
Stroke. 1972;3:449–455.
41. Jooss K, Yang YP, Wilson JM. Cyclophosphamide diminishes inflammation and prolongs transgene expression following delivery of adenoviral vectors to mouse liver and lung. Hum Gene Ther. 1996;7:1555–1566.[Medline] [Order article via Infotrieve]
42.
Kay
MA, Meuse L, Gown AM, Linsley P, Hollenbaugh D, Aruffo A, Ochs HD,
Wilson CB. Transient immunomodulation with anti-CD40 ligand antibody
and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene
transfer into mouse liver. Proc Natl Acad Sci
U S A. 1997;94:4686–4691.
43. Schiedner G, Morral N, Parks RJ, Wu Y, Koopmans SC, Langston C, Graha FL, Beaudet AL, Kochanek S. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat Genet. 1998;18:180–183.[Medline] [Order article via Infotrieve]
44.
Juul
R, Aakhus S, Bjönstad K, Gisvold SE, Blubakk AO, Edvinson L.
Calcitonin gene-related peptide (human 
-CGRP) counteracts
vasoconstriction in human subarachnoid haemorrhage. Neurosci
Lett. 1994;170:67–70.[Medline]
[Order article via Infotrieve]
45. Johnston FG, Bell BA, Robertson IJA, Miller JD, Haliburn C, O’Shaughnessy D, Riddel AJ, O’Laoire SA. Effect of calcitonin-gene-related peptide on postoperative neurological deficits after subarachnoid haemorrhage. Lancet. 1990;335:869–872.[Medline] [Order article via Infotrieve]
46. European CGRP in Subarachnoid Hemorrhage Group. Effect of calcitonin-gene-related-peptide in patients with delayed postoperative cerebral ischaemia after aneurysmal subarachnoid haemorrhage. Lancet. 1992;339:831–834.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  Y. Watanabe, F. M. Faraci, and D. D. Heistad Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2653 - H2658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Watanabe and D. D. Heistad Targeting cerebral arteries for gene therapy Exp Physiol, May 1, 2005; 90(3): 327 - 331. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Deng, T. J. Bivalacqua, N. N. Chattergoon, J. R. Jeter Jr., and P. J. Kadowitz Engineering Ex Vivo-Expanded Marrow Stromal Cells to Secrete Calcitonin Gene-Related Peptide Using Adenoviral Vector Stem Cells, December 1, 2004; 22(7): 1279 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yamaguchi, C. Zhou, D. D. Heistad, Y. Watanabe, and J. H. Zhang Gene Transfer of Extracellular Superoxide Dismutase Failed to Prevent Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage Stroke, November 1, 2004; 35(11): 2512 - 2517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cambj-Sapunar, M. Yu, D. R. Harder, and R. J. Roman Contribution of 5-Hydroxytryptamine1B Receptors and 20-Hydroxyeiscosatetraenoic Acid to Fall in Cerebral Blood Flow After Subarachnoid Hemorrhage Stroke, May 1, 2003; 34(5): 1269 - 1275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Watanabe, Y. Chu, J. J. Andresen, H. Nakane, F. M. Faraci, and D. D. Heistad Gene Transfer of Extracellular Superoxide Dismutase Reduces Cerebral Vasospasm After Subarachnoid Hemorrhage Stroke, February 1, 2003; 34(2): 434 - 440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. G. Khurana, L. A. Smith, T. A. Baker, D. Eguchi, T. O'Brien, and Z. S. Katusic Protective Vasomotor Effects of In Vivo Recombinant Endothelial Nitric Oxide Synthase Gene Expression in a Canine Model of Cerebral Vasospasm Stroke, March 1, 2002; 33(3): 782 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Didion, C. A. Hathaway, and F. M. Faraci Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1697 - H1703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Morishita Adventure of Gene Therapy Into the Brain: A New Era for Cardiovascular Gene Therapy Circ. Res., October 27, 2000; 87(9): 719 - 721. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||